MDM2 and its homolog MDMX are important regulators of the p53 tumor suppressor. MDMX overexpression occurs in a subset of human tumors and is an alternative mechanism for inactivating p53. MDMX is resistant to MDM2 inhibitors such as Nutlin, therefore MDMX overexpression may reduce the efficacy of MDM2 inhibitors currently entering clinical trials. Understanding the structure and regulation of MDMX will be critical for the successful targeting of p53 pathway in cancer therapy. MDMX does not have E3 ligase activity and its mechanism of p53 regulation is poorly understood. We showed that MDMX is an important target of signaling pathways that activate p53. DNA damage induces MDMX phosphorylation by ATM/Chk2, promotes MDMX nuclear translocation and degradation, and inhibits MDMX binding to CK1? and p53. To elucidate the mechanism of MDMX regulation and signal integration, we established a novel assay for the detection of protein intra-molecular interactions. We demonstrate that different MDMX domains engage in intra-molecular binding, which results in auto inhibition by p53 mimicry. We hypothesize that the intra-molecular interactions in MDMX are important for its function and regulation. The following experiments are proposed to further study the MDMX internal interactions during stress response. (1) Investigate the regulation of MDMX internal interactions using a novel proteolytic fragment release assay. (2) Determine the mechanism of p53 inhibition by MDMX and CK1?. (3) Investigate the mechanism of senescence stimulation by MDMX. (4) Determine the physiological functions of MDMX internal interactions using mouse model. These experiments should lead to a better understanding of the mechanisms that regulate MDMX and may identify novel strategy for targeting MDMX in human cancer.